Genetic analyses in a cohort of 191 pulmonary arterial hypertension patients

Pulmonary arterial hypertension (PAH) is a progressive and fatal disorder, which is diagnosed by a mean pulmonary artery pressure ≥ 25 mmHg at rest accompanied by a pulmonary artery wedge pressure (PAWP) ≤ 15 mmHg and a pulmonary vessel resistance (PVR) > 3 Wood units (WU), excluding other known causes of pulmonary hypertension, e.g., pulmonary diseases, thromboembolic diseases and left heart diseases [1]. Given the nonspecific clinical manifestations in the early stage, patients with PAH are frequently diagnosed late. Whenever the pulmonary pathologic changes become advanced and irreversible, prognosis and survival are poor and pessimistic. It has been reported that more than 20% patients have a two-year delay in the diagnosis of PAH [2], and if not treated timely, patients might progress into right heart failure [3] and the average survival duration after diagnosis is 2.8 years [4]. Idiopathic PAH (IPAH) corresponds to sporadic disease, without any familial history of PAH or known causes, which has an estimated prevalence of 5.9/1,000,000. Heritable PAH (HPAH) is diagnosed when there is a positive family history or when a pathogenic variant has been identified. For individuals with PAH, the most effective therapy to date is continuous injection of prostacyclin analogs. Other options include calcium channel blockers, oral prostanoids, phosphodiesterase type 5 inhibitors, and endothelin receptor antagonists [5]. Lung transplantation is effective, but long-term post-surgical survival is limited [6].

Since the 1990s, much has been learned about the molecular and genetic factors related to PAH. BMPR2 mutation is identified as the main genetic cause of PAH, accounting for 75%–90% of familial PAH [7, 8] and 3.5%–40% of sporadic cases [7, 9], with an autosomal dominant inheritance pattern [10]. The BMPR2 gene encodes bone morphogenetic protein receptor type II, which belongs to the transforming growth factor-β (TGF-β) superfamily and is involved in the regulation of cell growth and apoptosis. Two other genes, ACVRL1 [11] and ENG [12], which also encode receptors belonging to the TGF-β superfamily, have been more recently identified in PAH accompanying hereditary hemorrhagic telangiectasias (HHT). Similarly to BMPR2, these two genes affect vascular proliferation. With the development of genetics, more genes have been discovered to be implicated in the pathogenesis of PAH, including KCNK3 [13], CAV1 [14], SMAD9 [15], BMPR1B [16], but considerably less commonly so (1%–3%). The discovery of a causative mutation could help make an early diagnosis and provide an opportunity for family screening [17].

Pulmonary veno-occlusive disease (PVOD) is a rare disease that shares several clinical and hemodynamic similarities with IPAH but is distinct from IPAH in pathology and prognosis. An accurate diagnosis of PVOD in the early stage is crucial for suitable drug therapy and timely option for lung transplantation. Histological examination of lung biopsy is the gold standard for the diagnosis of PVOD, but it is often too invasive and unacceptable for the patient. Recent discoveries have indicated that gene mutations in eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) are responsible for inherited PVOD and pulmonary capillary hemangiomatosis (PCH) [18], and the 2015 ESC/ERS Guidelines declared that identification of a biallelic EIF2AK4 mutation was sufficient to make a diagnosis of PVOD/PCH without histological confirmation [1].

To identify the causal mutations in PAH patients and differentiate PVOD patients from those with IPAH, a custom gene panel targeting PAH and related diseases was used to test 191 probands with clinically suspected IPAH and HPAH. Furthermore, the performance of calling copy number variations (CNVs) from our panel data was evaluated.

A total of 191 patients were enrolled in our study and underwent genetic testing. Of the patients, 179 suffered from IPAH and 12 had HPAH. The main clinical findings are summarized in Table 1.

All samples were completed to perform high-quality genetic testing. Initially, a total of 119 PAH samples were submitted to single-gene testing of BMPR2 by Sanger sequencing. Later, we developed a PAH panel test in our lab. Then 97 samples with negative results in the BMPR2 test and 72 subsequently referred samples were then directed to a panel test involving 13 genes related to PAH. If the samples were still negative, MLPA testing was continued to search for the causative mutation (Additional file 1: Figure S1).

Sequencing of the 13 PAH genes (Additional file 1: Table S1) in the 169 probands yielded a mean depth of ~ 350× and coverage of 98.7%. Exons with low (< 20×) or no coverage were subjected to Sanger sequencing to obtain 100% coverage in the main gene, BMPR2. In addition, potential pathogenic mutations and rare variants of unknown significance (VUS) were confirmed using Sanger sequencing.

Causal mutations identified in the sequencing tests (both BMPR2 sequencing and panel sequencing), except for biallelic EIF2AK4, are described in Table 2. The table indicates that BMPR2 mutations were detected most frequently in 32 (17.9%) IPAH and 5 (41.7%) HPAH patients, while VUS are summarized in Additional file 1: Table S2. Notably, 4 HPAH families in our cohort indicated no suspected mutations either by panel sequencing or MLPA detection (Additional file 1: Figure S2), which required further genetic testing for the identification of new genes potentially related to PAH. In addition, in 6 patients clinically diagnosed with IPAH, homozygous or compound heterozygous EIF2AK4 mutations were detected (Table 3); thus, their diagnosis had to be corrected to PVOD/PCH.

Because deletion/duplication contributed considerably to BMPR2 pathogenic mutations in HPAH, we tried to detect CNVs in targeted NGS panel data. With the newly developed panelcn.MOPS pipeline by Povysil et al. [25], 12 BMPR2 deletions were detected, after excluding those flagged as low quality (Table 4). To confirm the results, MLPA was performed. Almost all BMPR2 CNVs were proved to be true, except for a slight inconsistency in patient PAH52, which panelcn.MOPS suggested only exon 11 del while MLPA revealed an exon 11–12 del in BMPR2, suggesting that the method implemented by panelcn.MOPS was highly effective for BMPR2 CNV detection. Confusingly, CNVs in ENG and ACVRL1 called by panelcn.MOPS were proved to be false (Additional file 1: Table S3), probably because of a high GC content (Additional file 1: Figure S3).

When trying to explore the genotype-phenotype correlation, we found that PAH patients with BMPR2 causal and rare mutations were younger at diagnosis (27.2y vs. 31.6y, p = 0.0003) and had more severe pulmonary hemodynamic impairment and a worse cardiac index (2.6 L/min/m² vs. 3.0 L/min/m², p = 0.0017) (Table 5) compared with those without BMPR2 mutations, in accord with previous reports [9]. In addition, patients with biallelic EIF2AK4 mutations had a lower diffusion capacity for carbon monoxide of lung (DLCO) than PAH patients (Additional file 1: Table S4).

To the best of our knowledge, this was the largest reported cohort of Chinese PAH patients undergoing genetic testing, which greatly contributed to expand PAH genetic database in Asian population. In the present study, 31.3% (56/179) IPAH and 66.7% (8/12) HPAH patients were detected causative mutations. Targeted NGS panel assay is a highly effective clinical tool, which allows not only for the detection of small sequence alterations, but also for an indication of CNVs with specific computational algorithms. Our results showed that 37 BMPR2 mutations (37/49, 75.5%) were identified in 32 IPAH and 5 HPAH by the sequencing methods and 12 BMPR2 CNVs (12/49, 24.5%) were indicated from the panel data and finally validated by MLPA, which was distinct from Cogan’s report [8] that a higher frequency of BMPR2 mutation was detected by deletions/duplications analysis in HPAH, compared with that by sequence analysis (48% vs. 37%). Therefore, an indication of CNVs from panel data would largely save costs in testing specified samples. Our results revealed that the newly developed and free available panelcn.MOPS pipeline [25] showed high sensitivity and specificity in exploring BMPR2 CNVs, ranging from single exons to the whole gene, and all were confirmed by MLPA, except for a slight inconsistency in which panelcn.MOPS suggested a single exon 11 del while MLPA revealed an exon 11–12 del in BMPR2 in patient PAH52. Conversely, CNVs detected in the genes ENG and ACVRL1 were all proved to be false, which might be related to a high GC content in these two genes.

Notably, 86 IPAH patients and 4 HPAH families were not detected any suspected mutations in the 13 known genes involved in our panel, indicating that other genes potentially responsible for the disease might exist. In most recent years, more genes were discovered, such as KLF2 [26], SOX17 [27], AQP1 [27]. We would add these novel genes and other candidate genes related to PAH into our panel in the future, such as FOXF1 [28], RASA1 [29], TBX4 [30], ABCA3 [31], SMAD1 [15]. Furthermore, we would also dedicate in novel gene discovering through whole exome sequencing (WES) later on, especially these four HPAH families with negative results in our cohort.

PVOD, featured with extensive and diffuse constriction and/or occlusion of pulmonary veins and venules, instead of pulmonary arteriopathy, shares several clinical and hemodynamic similarities with PAH, thus often leading to a misdiagnosis of IPAH. However, PVOD patients usually have a worse prognosis, and more importantly, the administration of PAH specific therapy (vasodilators) can precipitate severe acute pulmonary edema [32]. Hence, a diagnosis of PVOD/PCH distinct from that of PAH appears extremely important and identification of biallelic EIF2AK4 mutations makes it possible. In our study, in 6 patients clinically diagnosed with IPAH, homozygous or compound heterozygous EIF2AK4 mutations were detected; thus, their diagnosis should be corrected to PVOD.

Our clinical data demonstrated that patients with biallelic EIF2AK4 mutations had a lower DLCO value, suggesting a higher probability of biallelic EIF2AK4 detection in PAH patients with DLCO values < 40% of the predicted value. However, because of our limited PVOD sample size, the cut-off value of DLCO of predicted and the corresponding detection rate required further study.

It was reported that PVOD patients carrying biallelic EIF2AK4 mutations had a worse survival compared with IPAH patients [33] and PVOD patients without EIF2AK4 mutations [34], and showed no response to PAH therapy [34]. Inconsistently, neither of our two PVOD patients with intravenous injection of prostacyclin analogues, PAH3 and PAH151, demonstrated pulmonary edema. Regretfully, none of these 6 patients underwent microscopic examination of lung tissue; therefore, we were unaware of the differences that might have existed between PVOD patients who showed a good response to PAH-targeted drugs and those who did not.

With the extensive development of molecular genetics in clinical services, increasing amounts of genetic data have been obtained. Researchers are dedicated to exploring genotype-phenotype correlations in causative genes and PAH phenotypes. A meta-analysis of 1550 PAH patients revealed that PAH patients with BMPR2 mutations presented a younger age at diagnosis with more severe disease and a younger age at death or transplantation compared with non-carriers [9]. PAH patients with ACVRL1 mutations were younger at diagnosis and had a worse prognosis than other PAH patients [35]. In accord with previous reports, our data showed that PAH patients with BMPR2 mutations were younger at diagnosis and presented more severe pulmonary hemodynamic impairment and a worse cardiac index. Probably because of the limited sample size, we did not observe any differences between the ACVRL1 mutation group and other PAH patients (data not shown). The genotype-phenotype correlation exploring might help predict prognoses based on genetic results, and lay foundation for further mechanism study and target therapy.